If you're trying to quantify from band intensities from western blots, there are a number of pitfalls you have to deal with, and an appropriate loading control is only one of them. Ideally, I agree with Camille's answer; you're best bet is to normalize with respect to the total amount of protein loaded per lane, as there is no perfect 'houskeeping' protein you can use as a reference - things like actin, tubulin, GAPDH are not expressed consistently from cell to cell, especially after experimental perturbation. Furthermore, you have to be sure that your protein of interest an your loading control are being transferred from your gel to your membrane quantitatively (i.e. no blow-through, and no pile-up on the membrane... if you've got more than one layer of molecules on the blot, you can't detect them quantitatively). Then you have to be sure that both the protein of interest and the loading control are being detected quantitatively (i.e. that your primary and secondary antibodies are diluted such that they are not limiting, and that your detection method isn't over-saturating). The best way to determine if all your parameters are within their respective dynamic ranges is to load a 2x dilution series of your samples of interest on a blot, and run your detection protocol; hopefully there will be a range where both your protein of interest and your loading control shows a 2-fold change in signal intensity between lanes. Good luck.

We have always used beta-actin and alpha-tubulin and have had no difficulty. Mitochondrial proteins are tricky because the complement of mitochondria differs so much even from cell to cell.

you can use SNAP25, VAMP/synaptobrevin, syntaxin 1A/1B. the three proteins are known as SNARE proteins. VAMP/synaptobrevin is a 14 kDa vesicular protein which recognizes and associates to SNAP25 and syntaxin 1A (both expressed on the cytosolic side of plasma membrane) to form the so called SNARE complex. SNARE complex is the core of the neurotransmitter release apparatus at synapses

You can try actin related protein.

If you're trying to quantify from band intensities from western blots, there are a number of pitfalls you have to deal with, and an appropriate loading control is only one of them. Ideally, I agree with Camille's answer; you're best bet is to normalize with respect to the total amount of protein loaded per lane, as there is no perfect 'houskeeping' protein you can use as a reference - things like actin, tubulin, GAPDH are not expressed consistently from cell to cell, especially after experimental perturbation. Furthermore, you have to be sure that your protein of interest an your loading control are being transferred from your gel to your membrane quantitatively (i.e. no blow-through, and no pile-up on the membrane... if you've got more than one layer of molecules on the blot, you can't detect them quantitatively). Then you have to be sure that both the protein of interest and the loading control are being detected quantitatively (i.e. that your primary and secondary antibodies are diluted such that they are not limiting, and that your detection method isn't over-saturating). The best way to determine if all your parameters are within their respective dynamic ranges is to load a 2x dilution series of your samples of interest on a blot, and run your detection protocol; hopefully there will be a range where both your protein of interest and your loading control shows a 2-fold change in signal intensity between lanes. Good luck.

Hi Tomasz,

I completely agree with Camille and I think that the best suggestion is from Bryan. Coupled together, you will verify your protein load and take into account the potential variations during the blotting procedure.Furthermore, you will have Two point of value to compare in your quantification process.

I would like to add a comment on your revelation step. Make sure that your results will give data that could be interpreted. I often prefer Fluorescence for the dynamic range of the quantification for example.

If you want to have a set of pictures for a paper with an internal control try some of the one suggested by Marco and peroxiredoxin 2...but do not make your quantification with regard to them, differenciation of neuronal cells or related one induced a strong change ine the metabolic pathways so that lots of house keeping proteins are also affected by these changes.

Good luck

These answers are all very good, and I only have one thing to add. I normally quantify total protein using a Bradford kit. This ensures equal loading of total protein from the beginning of your experiment. Then, you can check "housekeeping" genes to see which of them is expressed consistently across all lanes, and this will give you a good idea of the best loading control. This is important especially if you are looking at different stages of differentiation on one gel.

Good luck!

In addition to checking the total protein content in each well using Ponceau or a similar method as mentioned above, it would be helpful (if you don't already do this) to protein-normalize your samples before you load them as Biliana suggested. I use BCA for this purpose, but Bradford works just as well. By ensuring that you are loading the same amount of lysate in each well, you make the interpretation of your results easier. Then the Ponceau or housekeeping protein is really just to double-check your calculations and account for pipetting error.

I find that it is really critical to get a good estimate of the protein concentration to begin with. The three main methods out there are:

Absorbance at 280nm (requires a UV transparent micro-plate or quartz cuvettes)

Bradford (Detergents like SDS can affect results)

BCA (redox active reagents (eg beta-mercaptoethanol can affect results)

I use a modified version of the bradford assay that is linear and more sensitive than the traditional method. The only difference between this and the traditional method is that you have to measure absorbance at 450nm in addition to 585nm and then use the absorbance ratio (585/450 instead of just the absorbance at 585nm) for your calculations.

Here is a nice video to explain the protocol in detail:

J Vis Exp. 2010; (38): 1918.

http://www.jove.com/video/1918/linearization-of-the-bradford-protein-assay?ID=1918

Once you have really even loading, you can work on optimizing other conditions (eg. mass of protein loaded in each well) until you get a quantitative blot (ie. you get 2X from densitometry when 2X the protein mass was loaded).

If you want to compare differentiated cells to progenitors. You will need to find a loading control that has similar expression levels before and after differentiation.

It seems logical that neurons will have more actin and tubulin than neural progenitor cells because they have neurites. However, I've seen many papers use these as loading controls for similar experiments. Maybe a nuclear protein like TATA-binding protein (TBP) or Lamin would be better. You can determine this yourself as long as you have even loading.

I also recommend you do the Ponceau stain. This is a good way to check if your loading is even, but I don't use this to normalize my data because I cannot get quantitative densitometry on this signal with the scanner in my lab.

Try nestin may be

We use beta actin

Thank you all for the answers. As I thought a good loading control protein is hard to find for my particular experiment and of course I agree with dr Bryan Crawford that chemiluminescence Western should not be treated as a quantitative assay. I think that Ponceau normalisation will be the best for "semi - quantifying" and I think I will try lamin visalisation as a reference loading control for preparing a photo for publication.

Johnathan - I also use ratiometric Bradford - the curve (sensitivity) is better than in standard Bradford - it is very good method but in my experiments it only works for lysate concentration between 2-7 ug but this is probably the matter of buffer composition and dilution. Unfortunetely when I prepare lysates in Laemly buffer with SDS and B-mercapthoethanol (for hydrophobic mitochondrial proteins) this method cannot be used.

Didier - you have suggested using peroxiredoxin - 2 as internal control. Is the expression of this protein unchanged during neuronal differantiation ? Have you got any experience with that?

To all - When I will have a photo of Ponceau staining what free program can be used to measure and compare all bands between the lines?

Thank you once again and best in your research!

True, everything changes. We usually use actin

There is no single loading control that will work across developmental time points. Most proteins will be differentially expressed a function of development (i.e. time/lifespan). Hence, you should probably use a total protein loading control. Ponceau is problematic so I would suggest Memcode (as suggested earlier) or amido black; however, amido black must be used after you visualize your protein so in theory your primary/secondary antibody could contribute to your "total" protein signal.

Actin is a poor loading control in almost all cases and it is generally preferred because it is stable from lane to lane. That's a problem though; what you want is a signal that reflects how much protein has actually been loaded, not a loading control that simply gives you a pretty blot (http://www.ncbi.nlm.nih.gov/pubmed/16688701).

For this experiment I think you have to use a total protein stain, this paper provides a good discussion:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567873/

Hope that helps.

Chris

Reversible protein stains will help. Have you considered using tyrosine hydroxylase as an additional (not sole) context since you are looking at dopaminergic differentiation?

Chris raises an important concern.

Hi Tomasz,

In a 2D-DIGE experiment, we have seen that one of the Housekeeping proteins less affected with neuropathological process was Peroxiredoxine-2. A colleague use this target with neuronal cell lines in experimental protocols that are not differnetciation, but showing no strong alteration of the peroxiredoxin expression and high variation of actin and tubulin. We are now using this target mainly to draw a significant picture for communication or publication as a "visual control" . Nevertheless, all the data are obtained with a ponceau red validation of the loading. But I will try the Memcode as suggested by others.

For a Free application to quantify your blots you can try the well known Image J that could be downoaded at:

http://rsbweb.nih.gov/ij/download.html

Have a good day

Didier

I will try the Memcode - thanks! I use the well documented protocol for differentiation of LUHMES cells so I do not check for tyrosine hydroxylase espression and other markers of dopaminergic differentiation all the time:

http://www.ncbi.nlm.nih.gov/pubmed/21434924

It is a very user friendly cell line - the only problem is that you have to use coated plastic and they grow only on Nunclon delta surface but it is much easier to obtain differentiated cells from LUHMES than in case of SH-SY5Y neuroblastomas that i used before. I really advertise this cell line for neuronal studies if you do not want to use stem cells.

Have a good weekend

Tomek